1. Field of the Invention
This invention relates to an amino acid sequence of a precursor polypeptide of phytosulfokine. Phytosulfokine is a peptide known to enhance proliferation of a plant cell. This invention also relates to a gene encoding said precursor polypeptide. Moreover, this invention relates to a method to promote proliferation of a plant cell by incorporation of said gene into a plant.
2. Description of Related Art
The progression of plant gene engineering techniques has enhanced the production of transgenic plants incorporating various exogenous genes. Such techniques are available for various plant species and play an important role in the development of industry. For example, such techniques enable the production of novel plant species with improved productivity of their secondary metabolites.
For the production of a transgenic plant, it is necessary to cultivate a small number of transformed cells, wherein a certain exogenous gene is incorporated, to regenerate a plant body. However, in the case of a plant cell wherein a certain exogenous gene is incorporated, proliferation of such a cell is very slow. Therefore, regeneration of a plantlet to produce a transgenic plant may be difficult. A plant cell secretes unknown growth factors into the extracellular medium to promote cell division. However, when the plant cells exist in lower density clusters than necessary, proliferation of the plant cells becomes difficult. Often it takes too much time for the growth factor to reach sufficient concentration or the degradation rate of the growth factor exceeds that of secretion. Moreover, in many plant species, cell culture itself is difficult or the rate of cell proliferation is very slow. Therefore, the development of a technique for the promotion of plant cell proliferation has been desired.
The inventors have purified and isolated phytosulfokine (PSK), as a peptidyl plant growth factor described above (Y. Matsubayashi and Y. Sakagami, Proc. Natl. Acad. Sci. USA 93, p7623, 1996). PSK is one of the plant growth factors contained in so-called xe2x80x9cconditioned medium: CMxe2x80x9d, a medium once used for cell culture. It is known that the PSK is secreted into the extra-cellular medium and functions as an autocrine factor. It is also known that the tyrosine residue of PSK is sulfated by post-translational modification. Two types of PSK, namely PSK-xcex1 and PSK-xcex2, are recognized and these sequences are described below. PSK-xcex2 is an enzymatic degradation product of PSK-xcex1 and the cell proliferation activity observed in PSK-xcex2 is less than one-tenth of that of PSK-xcex1.
PSK-xcex1: Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln
PSK-xcex2: Tyr(SO3H)-Ile-Tyr(SO3H)-Thr
The existence of a physiologically active plant peptide or protein in which the tyrosine residue is sulfated was not known until the discovery of PSK. In animals, about 30 species, including cholecystokinin or gastrin, are known to exhibit such a property. They are all extra-cellularly secreted peptides bio-synthesized as their precursors, sulfated and processed during transition via the trans-Golgi network. It is assumed that excision of the PSK sequence occurs in the same manner. The presence of a certain signal sequence is predicted in the precursor peptides wherein their tyrosine residue is specifically sulfated, as the precursor peptides are secreted into the extra-cellular region. Concerning the PSK peptide, the existence of a precursor peptide is predicted based on such knowledge. Therefore, the gene encoding the precursor polypeptide of rice PSK was isolated and the base sequence of the precursor gene was determined. The gene thus obtained was incorporated into rice Oc culture cells, and the effect of the gene was investigated. The over-expression of the gene encoding the PSK precursor polypeptide increased the secretion of PSK into cultured medium and promoted the proliferation of rice Oc cells.
The precursor polypeptide of PSK (preprophytosulfokine) of this invention is identified by an amino acid sequence referred to as SEQ ID NO: 1. The gene encoding preprophytosulfokine is identified by a base sequence referred to as SEQ ID NO: 2.
In general, one amino acid is encoded by a plurality of base sequences of DNA. Therefore, a plurality of genes, other than native gene of this invention, might encode amino acid sequences identical to preprophytosulfokine. The gene of this invention is not to be limited to only the native gene and is intended to include many other base sequences encoding preprophytosulfokine.
The precursor polypeptide of PSK of this invention includes a polypeptide having an amino acid sequence at least 40% sequence homology to the amino acid sequence of SEQ ID NO: 1, while retaining the biochemical characteristics of preprophytosulfokine. In a preferred form, the precursor polypeptide of this invention has more than 50% sequence homology to the amino acid sequence of SEQ ID NO: 1. In a more preferred form, the precursor polypeptide of this invention has more than 80% sequence homology to the amino acid sequence of SEQ ID NO: 1.
Moreover, the gene of this invention includes a gene that encodes the precursor polypeptide of PSK described above consisting of a base sequence that hybridizes with the base sequence of SEQ ID NO: 2 under stringent conditions.
The proliferation of a plant cell can be promoted by the incorporation of a gene encoding preprophytosulfokine. A plant cell, wherein a certain exogenous gene is incorporated, tends to decrease its proliferation rate. Especially, this technique enhances the proliferation of said cell with a decreased proliferation rate. Therefore, cell differentiation and regeneration of a plantlet would be achieved by incorporation of the gene encoding preprophytosulfokine. Moreover, growth enhancement of a plant would be achieved by incorporation of the gene. The gene disclosed in this invention can be incorporated into various plants. The plants described below are plants preferable for incorporation of the gene. These are monocotyl plants such as rice, maize, asparagus and wheat or dicotyl plants such as arabidopsis, tobacco, carrot, soy bean, tomato and potato. For a technique to produce a transformant, ordinary techniques known in the art can be adopted. For an example of an available vector, pAct-nos/Hmz can be mentioned. Such a vector can be introduced into Agrobacterium, for example, and a transformant can be produced by infection of a callus or an infant plant. The Examples described above and the embodiments described below are preferred embodiments of this invention and do not intend to exhibit the limits or the range of this invention.
These and other objects and advantages of the invention will become more apparent upon a reading of the detailed description and drawings.